PROJECT SUMMARY/ABSTRACT Chemotherapy has been the centerpiece of cancer treatment in modern society, and will remain so for the foreseeable future. DNA-damaging compounds have seen the most use historically in this regard, due to cancer cells? increased rates of growth and proliferation relative to normal, healthy cells. However, these treatments often cause off-target effects and resistance development. Therefore, compounds that can damage cancer cell DNA in a selective fashion and with a mechanism complementary to current clinical treatments offer solutions to both of these issues. The natural product leinamycin (LNM) is a DNA-damaging compound that only elicits its DNA-damaging effects upon activation by thiols, and has nanomolar activity against tumor cells resistant to clinically utilized drugs. LNM E1 is an engineered LNM analog that upon activation by reactive oxygen species (ROS) exerts its antitumor activity via a mechanism orthogonal to LNM. A single scaffold that can be primed for DNA damage in two complementary ways is unique to this set of compounds, and sets the stage for further optimization of the LNM scaffold in terms of anticancer activity. The biosynthetic origin of LNM in Streptomyces has been an area of intense study, and many of the biosynthetic steps have been elucidated and led to discoveries involving unprecedented chemical steps. The long-term goals of this project are to harness the power of a mechanistic understanding of LNM biosynthesis to genetically alter producing organisms for production of analogs with improved therapeutic properties. This proposal contains two aims: (i) investigate the mechanism of a key biosynthetic step and (ii) access rationally-designed analogs of both LNM and LNM E1 to answer specific questions about the compounds? biological activities. The central hypothesis of this proposal is that LNM compounds can be tuned and improved in both stability and potency through rational design. This hypothesis is rationalized by Nature?s ability to produce an array of LNM-type compounds, which have served as the inspiration for the structural changes proposed herein. The outcomes of this application will be a mechanistic understanding of one of the key steps of LNM biosynthesis and access to novel LNM analogs that can be used to answer key questions about LNM DNA-damaging and anticancer activity. These findings can be applied to the long-term goal of rational biosynthetic manipulation to produce targeted structural LNM analogs for further anticancer development.